In captivity, neotropical Seba's short-tailed bats (Carollia perspicillata) (Chiroptera: Phyllostomidae) reproduce readily, and contraception can constitute an opportunity to prevent uncontrolled reproduction, which can lead to overpopulation as well as inbreeding. The authors developed a safe and efficient work flow, anesthetic, and surgical castration protocol for the reproductive control of a captive population of more than 500 bats housed at the rainforest house at the Vienna Zoo, Austria. An anesthetic protocol using 0.003 mg medetomidine per bat (approximately 0.18 mg/kg) and 0.3 mg ketamine per bat (approximately 18 mg/kg) achieved surgical anesthesia in 323/328 bats, with the remaining five requiring supplementation with isoflurane. A single bat, which had a pre-existing underlying infection, died during anesthetic induction. Standard closed orchiectomy was performed on a total of 336 bats. The practicable and safe working protocol developed required three veterinarians and 3–4 assistants for the orchiectomy of this large group of captive bats. With the described work flow, the period for castration averaged 2.38 min/bat, resulting in managing 28.2 bats/h.